IIa. Determinants of gastrin-releasing peptide receptor (GRP-R) high affinity for agonists. Three of the 4 bombesin (Bn) receptors [GRP-R, NMB-R and BB-4) bind Bn with high affinity whereas the orphan receptor, BRS-3, which has 50% homology with other 3 Bn receptors, does not. This difference was used to determine residues important for high affinity for Bn. BRS-3-R substitution in GRP-R of R288H, Q121R, P199S and A308S caused a significant decrease in receptor affinity for Bn. The analogous substitutions in BRS-3 (H294R, R127Q, S205P, S315A) caused a 100-fold gain in affinity. A preliminary map of some of amino acids comprising the agonist binding packet for GRP-R was proposed. IIb. Ligand-induced internalization of cholecystokinin receptors (CCK-R). The carboxyl terminal has been shown to influence internalization of some G protein-coupled receptors. To investigate this COOH terminal mutants of the CCKB receptor and CCKA receptors were made. The COOH terminal truncated CCKA-R, but not that of the CCKB-R, internalized ligand normally, despite both having normal G-protein coupling and receptor affinity. The abnormal internalization of the CCKB-R was shown due to loss of serines and threonines in the COOH terminus. These results shown that despite a 50% homology between the CCKA-R and CCKB-R, the structural determinants that mediate the endocytic pathways of these 2 receptors reside in different receptor regions. IIc. Visualization of G protein-coupled receptor trafficking with the aid of green fluorescent protein. A chimeric protein consisting of the CCKA receptor (CCKA-R) and the green fluorescent protein (GFP) was used to study receptor localization, internalization, and recycling in 4 different cell lines. The chimeric receptors functioned normally. The receptor underwent ligand-dependent internalization in 3 cell lines and constitutive in a 4th suggesting cell-specific regulation of receptor internalization. The kinetics of receptor internalization and recycling were determined. These studies demonstrate the usefulness of this technique to study receptor regulation and trafficking.